Transport of sulfobromophthalein and taurocholate in the HepG2 cell line in relation to the expression of membrane carrier proteins.

The transport of two different classes of organic anions (cholephilic dyes; the sulfobromophthalein, BSP, and bile acids; taurocholate, TC) was investigated in the HepG2 cell line. At 37 degrees C, BSP uptake was found to be biphasic with an apparent saturative curve in the concentration range between 0-6 microM followed by a linear component up to 18 microM. Kinetic constant determination showed an apparent Km of 26.6 +/- 3.1 microM and a Vmax of 5.64 +/- 0.82 nmol BSP.min-1.mg prot-1. At 4 degrees C, uptake was linear. By subtracting this latter component from the total uptake, a saturable, carrier mediated uptake was found with an apparent Km of 3.6 +/- 1.0 microM BSP and a Vmax of 0.37 +/- 0.04 nmol BSP.min-1.mg prot-1 (m +/- SEM, n = 6). These values were fully comparable with those found in freshly isolated male hepatocyte. Immunoblot analysis of HepG2 cell plasma membrane revealed the presence of bilitranslocase when tested against a monospecific antibody against this carrier molecule. On the contrary, TC uptake was linear up to concentration of 100 microM TC. No difference was observed in the presence or absence of Na+. Immunoprecipitation analysis showed the absence of the putative carrier of TC. These data indicate that the HepG2 cell line expresses a functioning bilitranslocase-mediated system. Conversely, carrier mediated bile acid uptake is absent in line with the lack of expression of the carrier protein.     

Cloning and Expression of a Hexose Transporter Gene Expressed during the Ripening of Grape Berry

The ripening of grape (Vitis vinifera L.) is characterized by massive sugar import into the berries. The events triggering this process and the pathways of assimilate transport are still poorly known. A genomic clone Vvht1 (Vitis vinifera hexose transporter1) and the corresponding cDNA encoding a hexose transporter whose expression is induced during berry ripening have been isolated. Vvht1 is expressed mainly in the berries, with a first peak of expression at anthesis, and a second peak about 5 weeks after véraison (a viniculture term for the inception of ripening). Vvht is strictly conserved between two grape cultivars (Pinot Noir and Ugni-Blanc). The organization of the Vvht1 genomic sequence is homologous to that of the Arabidopsis hexose transporter, but differs strongly from that of the Chlorella kessleri hexose transporter genes. The Vvht1 promoter sequence contains several potential regulating cis elements, including ethylene-, abscisic acid-, and sugar-responsive boxes. Comparison of the Vvht1 promoter with the promoter of grape alcohol dehydrogenase, which is expressed at the same time during ripening, also allowed the identification of a 15-bp consensus sequence, which suggests a possible co-regulation of the expression of these genes. The expression of Vvht1 during ripening indicates that sucrose is at least partially cleaved before uptake into the flesh cells.     

Changes in cell-wall polysaccharides from the mesocarp of grape berries during veraison

Softening of grape berries ( Vitis vinifera L. × V. labruscana cv. Kyoho) was evaluated by studying changes in composition and degradation of cell-wall polysaccharides. The grape berry softens at the beginning of the second growth cycle many weeks before harvest. The softening stage is called 'veraison' by viticulturists. On day 50 after full bloom, green hard berries (before veraison [BV]), softening berries (veraison [V]) and partly peel colored berries (C) were selected from the same clusters. In addition, mature berries (M) were collected on day 78 after full bloom. Mesocarp tissues at each stage were fractionated into hot water-soluble (WS), hot EDTA-soluble (pectin), alkali-soluble (hemicellulose) and residual (cellulose) fractions. Neutral and acidic sugar contents of WS and pectin fractions decreased only after the V stage, while the neutral sugar content of the hemicellulose fraction decreased from the BV to V stages. Cellulose content constantly decreased as the berry ripened, but the large decrease was found from the BV to V stages. Molecular masses of pectic and hemicellulosic polysaccharides decreased from the BV to V stages. Hemicellulosic xyloglucan was markedly depolymerized from the BV to V stages. The neutral and acidic sugar composition of each fraction changed little during the berry ripening. These data indicated that softening of berry during veraison involved the depolymerization of pectin and xyloglucan molecules and decrease in the amounts of hemicellulose and cellulose.     

Expression and In Situ Localization of Two Major PR Proteins of Grapevine Berries during Development and after UV-C Exposition

In grapevine Vitis vinifera L. cv Pinot noir, the Pathogenesis-Related (PR) proteins CHI4D and TL3 are among the most abundant extractable PR proteins of ripe berries and accumulate during berry ripening from véraison until full maturation. Evidence was supplied in favor of the involvement of these two protein families in plant defense mechanisms and plant development. In order to better understand CHI4D and TL3 function in grapevine, we analyzed their temporal and spatial pattern of expression during maturation and after an abiotic stress (UV-C) by in situ hybridization (ISH) and immunohistolocalization. In ripening berries, CHI4D and TL3 genes were mainly expressed in the exocarp and around vascular bundles of the mesocarp. In UV-C exposed berries, CHI4D and TL3 gene expression was strongly induced before véraison. Corresponding proteins localized in the exocarp and, to a lesser extent, around vascular bundles of the mesocarp. The spatial and temporal accumulation of the two PR proteins during berry maturation and after an abiotic stress is discussed in relation to their putative roles in plant defense.     

ABA and GA3 increase carbon allocation in different organs of grapevine plants by inducing accumulation of non‐structural carbohydrates in leaves,enhancement of phloem area and expression of sugar transporters

Grape quality for winemaking depends on sugar accumulation and metabolism in berries. Abscisic acid (ABA) and gibberellins (GAs) have been reported to control sugar allocation in economically important crops, although the mechanisms involved are still unknown. The present study tested if ABA and gibberellin A₃ (GA₃) enhance carbon allocation in fruits of grapevines by modifying phloem loading, phloem area and expression of sugar transporters in leaves and berries. Pot‐grown Vitis vinifera cv. Malbec plants were sprayed with ABA and GA₃ solutions. The amount of soluble sugars in leaves and berries related to photosynthesis were examined at three points of berry growth: pre‐veraison, full veraison and post‐veraison. Starch levels and amylase activity in leaves, gene expression of sugar transporters in leaves and berries and phloem anatomy were examined at full veraison. Accumulation of glucose and fructose in berries was hastened in ABA‐treated plants at the stage of full veraison, which was correlated with enhancement of Vitis vinifera HEXOSE TRANSPORTER 2 (VvHT2) and Vitis vinifera HEXOSE TRANSPORTER 6 (VvHT6) gene expression, increases of phloem area and sucrose content in leaves. On the other hand, GA₃ increased the quantity of photoassimilates delivered to the stem thus increasing xylem growth. In conclusion, stimulation of sugar transport by ABA and GA₃ to berries and stems, respectively, was due to build‐up of non‐structural carbohydrates in leaves, modifications in phloem tissue and modulation in gene expression of sugar transporters.     

Treatment of Grape Berries,a Nonclimacteric Fruit with a Synthetic Auxin,Retards Ripening and Alters the Expression of Developmentally Regulated Genes

Treatment of grape (Vitis vinifera L.) berries with the synthetic auxin-like compound benzothiazole-2-oxyacetic acid (BTOA) caused a delay in the onset of ripening of approximately 2 weeks. This was manifested as a retardation of the increases in berry weight, color, deformability, and hexose concentration. BTOA treatment also delayed by 2 weeks the increase in abscisic acid level that normally accompanies ripening and altered the expression of a number of developmentally regulated genes. A putative vacuolar invertase, which is normally expressed from berry set until ripening and turned off after ripening commences, remained expressed throughout development in BTOA-treated grape berries. This elevated expression resulted in increased levels of invertase activity. In contrast, the up-regulation of four other genes normally switched on at the time of ripening was delayed in BTOA-treated fruit. These included chalcone synthase and UDP-glucose-flavonoid 3-O-glucosyl transferase, both of which are involved in anthocyanin synthesis, a chitinase, and a ripening-related gene of an unknown function. These observations support the view that auxins (perhaps in conjunction with abscisic acid) may have a role in the control of grape berry ripening by affecting the expression of genes involved in the ripening process.     

The kinetics of sulfobromophthalein uptake by rat liver sinusoidal vesicles

The kinetics of bromo[35S]sulfophthalein (35S-BSP) binding by and uptake across the hepatocyte sinusoidal membrane were investigated using isolated rat liver sinusoidal membrane vesicles containing K+ as the principal internal inorganic cation. Uptake of 35S-BSP into vesicles was found to be temperature dependent, with maximum uptake between 35 and 40 degrees C; only binding occurred at or below 15 degrees C. Uptake at 37 degrees C was saturable and resolvable by Eadee-Hofstee analysis into two components: one with high affinity (Km = 53.1 microM) but low capacity, and the second of low affinity (Km = 1150 microM) but high capacity. By pre- or post-incubation, respectively, with unlabelled BSP, trans-stimulation and counter transport of 35S-BSP could also be demonstrated in these vesicles. Uptake was inhibited competitively using 5 microM Rose bengal and 10 microM indocyanine green, and non-competitively using 10 microM DIDS. Taurocholate did not inhibit uptake, and actually enhanced transport at concentrations greater than or equal to 250 microM. Imposition of inwardly directed inorganic ion gradients resulted in the enhancement of 35S-BSP transport when chloride ions were part of this gradient, irrespective of the cation employed whereas there was no apparent cation effect. However, substitution of 10 mM Na+ for 10 mM K+ as the internal cation resulted in a significant increase in uptake in the presence of external K+ as compared to Na+ gradients. This effect was not observed when 10 mM Tris+ was employed as the internal cation. The kinetics of 35S-BSP uptake by isolated sinusoidal membrane vesicles are indicative of facilitated transport. While the observed inorganic ion effects suggest a possible electrogenic component, the driving forces for hepatic BSP uptake remain uncertain. Isolated sinusoidal membrane vesicles provide a useful technique for studying hepatic uptake processes independent of circulatory or subsequent cellular phenomena.     

Transporters expressed during grape berry (Vitis vinifera L.) development are associated with an increase in berry size and berry potassium accumulation

Potassium accumulation is essential for grapevine (Vitis vinifera L.) growth and development, but excessive levels in berries at harvest may reduce wine quality particularly for red wines. In addition to decreasing the free acid levels, potassium also combines with tartaric acid to form largely insoluble potassium bitartrate. This precipitates during winemaking and storage, resulting in an increase in wine pH that is associated with negative impacts on wine colour, flavour, and microbiological stability. For these reasons, a better understanding of potassium transport and accumulation within the vine and berries is important for producing fruit with improved winemaking characteristics. Here two genes encoding KUP/KT/HAK-type potassium transporters that are expressed in grape berries are described. Their function as potassium transporters was demonstrated by complementation of an Escherichia coli mutant. The two transporters are expressed most highly in the berry skin during the first phase of berry development (pre-veraison), with similar patterns in two grapevine varieties. The timing and location of expression of these transporters are consistent with an involvement in potassium accumulation in grape berries.     

Sugar accumulation in grape berries. Cloning of two putative vacuolar invertase cDNAs and their expression in grapevine tissues.

During grape berry (Vitis vinifera L.) ripening, sucrose transported from the leaves is accumulated in the berry vacuoles as glucose and fructose. To study the involvement of invertase in grape berry ripening, we have cloned two cDNAs (GIN1 and GIN2) from berries. The cDNAs encode translation products that are 62% identical to each other and both appear to be vacuolar forms of invertase. Both genes are expressed in a variety of tissues, including berries, leaves, roots, seeds, and flowers, but the two genes have distinct patterns of expression. In grape berries, hexose accumulation began 8 weeks postflowering and continued until the fruit was ripe at 16 weeks. Invertase activity increased from flowering, was maximal 8 weeks postflowering, and remained constant on a per berry basis throughout ripening. Expression of GIN1 and GIN2 in berries, which was high early in berry development, declined greatly at the commencement of hexose accumulation. The results suggest that although vacuolar invertases are involved in hexose accumulation in grape berries, the expression of the genes and the synthesis of the enzymes precedes the onset of hexose accumulation by some weeks, so other mechanisms must be involved in regulating this process.     

Grapevine aquaporins: gating of a tonoplast intrinsic protein (TIP2;1) by cytosolic pH

Grapevine (Vitis vinifera L.) is one of the oldest and most important perennial crops being considered as a fruit ligneous tree model system in which the water status appears crucial for high fruit and wine quality, controlling productivity and alcohol level. V. vinifera genome contains 28 genes coding for aquaporins, which acting in a concerted and regulated manner appear relevant for plant withstanding extremely unfavorable drought conditions essential for the quality of berries and wine. Several Vv aquaporins have been reported to be expressed in roots, shoots, berries and leaves with clear cultivar differences in their expression level, making their in vivo biochemical characterization a difficult task. In this work V. vinifera cv. Touriga nacional VvTnPIP1;1, VvTnPIP2;2 and VvTnTIP2;1 were expressed in yeast and water transport activity was characterized in intact cells of the transformants. The three aquaporins were localized in the yeast plasma membrane but only VvTnTIP2;1 expression enhanced the water permeability with a concomitant decrease of the activation energy of water transport. Acidification of yeast cytosol resulted in loss of VvTnTIP2;1 activity. Sequence analysis revealed the presence of a His(131) residue, unusual in TIPs. By site directed mutagenesis, replacement of this residue by aspartic acid or alanine resulted in loss of pH(in) dependence while replacement by lysine resulted in total loss of activity. In addition to characterization of VvTn aquaporins, these results shed light on the gating of a specific tonoplast aquaporin by cytosolic pH.     

Characterization of mitochondrial dicarboxylate/tricarboxylate transporters from grape berries

Grape berries (Vitis vinifera L fruit) exhibit a double-sigmoid pattern of development that results from two successive periods of vacuolar swelling during which the nature of accumulated solutes changes significantly. Throughout the first period, called green or herbaceous stage, berries accumulate high levels of organic acids, mainly malate and tartrate. At the cellular level fruit acidity comprises both metabolism and vacuolar storage. Malic acid compartmentation is critical for optimal functioning of cytosolic enzymes. Therefore, the identification and characterization of the carriers involved in malate transport across sub-cellular compartments is of great importance. The decrease in acid content during grape berry ripening has been mainly associated to mitochondrial malate oxidation. However, no Vitis vinifera mitochondrial carrier involved in malate transport has been reported to date. Here we describe the identification of three V. vinifera mitochondrial dicarboxylate/tricarboxylate carriers (VvDTC1-3) putatively involved in mitochondrial malate, citrate and other di/tricarboxylates transport. The three VvDTCs are very similar, sharing a percentage of identical residues of at least 83 %. Expression analysis of the encoding VvDTC genes in grape berries shows that they are differentially regulated exhibiting a developmental pattern of expression. The simultaneous high expression of both VvDTC2 and VvDTC3 in grape berry mesocarp close to the onset of ripening suggests that these carriers might be involved in the transport of malate into mitochondria.     

Characterization, induction by wounding and salicylic acid, and activity against Botrytis cinerea of chitinases and β‐1,3‐glucanases of ripening grape berries

In order to better understand the defense strategy of grape berries ( Vitis vinifera L. cv. Pinot noir) as they mature, the activities of the defense‐related proteins, chitinase (CHV, EC 3.2.1.14) and β‐1,3‐glucanase (laminarinase, EC 3.2.1.39) were first estimated in berries at different maturation stages. Chitinase levels rose proportionally to the berry reducing sugar content, an indicator of the berry ripening degree, up to values 10 times higher than the ones seen in resting grapevine leaves. This rise in activity was due to the accumulation of two isoforms, CHV 5 and CHV 11. One more chitinase isoform, CHV 12, appeared in senescent berries. Conversely, no glucanase activity could be detected in berries at any maturation stage. Accumulation of chitinases and (β‐1,3‐glucanases could be stimulated by wounding the berry peduncle. Adding salicylic acid to the wounded berries only potentiated the wounding effect on the berry chitinase activity. The most active chitinase isoform, CHV 5, was purified to homogeneity. It represented about 40% of the total extractable protein content of a ripe berry. Its molecular mass was estimated to be 31 kDa. The peptide sequencing of four of its tryptic fragments revealed strong homologies to several class IV chitinases. Finally, it was shown to inhibit the germination of conidia of Botrytis cinerea by 50% at a concentration of 7.5 µg ml⁻¹.     

Characterization and identification of steroid sulfate transporters of human placenta

Human trophoblasts depend on the supply of external precursors, such as dehydroepiandrosterone-3-sulfate (DHEA-S) and 16 alpha-OH-DHEA-S, for synthesis of estrogens. The aim of the present study was to characterize the uptake of DHEA-S by isolated mononucleated trophoblasts (MT) and to identify the involved transporter polypeptides. The kinetic analysis of DHEA-(35)S uptake by MT revealed a saturable uptake mechanism (K(m) = 26 microM, V(max) = 428 pmol x mg protein(-1) x min(-1)), which was superimposed by a nonsaturable uptake mechanism (diffusion constant = 1.2 microl x mg protein(-1) x min(-1)). Uptake of [(3)H]DHEA-S by MT was Na(+) dependent and inhibited by sulfobromophthalein (BSP), steroid sulfates, and probenecid, but not by steroid glucuronides, unconjugated steroids, conjugated bile acids, ouabain, p-aminohippurate (PAH), and bumetanide. MT took up [(35)S]BSP, [(3)H]estrone-sulfate, but not (3)H-labeled ouabain, estradiol-17beta-glucuronide, taurocholate, and PAH. RT-PCR revealed that the organic anion-transporting polypeptides OATP-B, -D, -E, and the organic anion transporter OAT-4 are highly expressed, and that OATP-A, -C, -8, OAT-3, and Na(+)-taurocholate cotransporting polypeptide (NTCP) are not or are only lowly expressed in term placental tissue and freshly isolated and cultured trophoblasts. Immunohistochemistry of first- and third-trimester placenta detected OAT-4 on cytotrophoblast membranes and at the basal surface of the syncytiotrophoblast. Our results indicate that uptake of steroid sulfates by isolated MT is mediated by OATP-B and OAT-4 and suggest a physiological role of both carrier proteins in placental uptake of fetal-derived steroid sulfates.     

Proteomic analysis of β-1,3-glucanase in grape berry tissues

Grape berries are considered recalcitrant materials in proteomic analysis, because berry tissues contain large amounts of secondary metabolites, especially phenolic compounds, which severely interfere with protein extraction and electrophoresis separation. We report hereby a PVPP/TCA-based protein extraction protocol for grape berries. Phenolic compounds in berry extracts were removed with repeated PVPP cleanups, and proteins were recovered with TCA precipitation. Protein resolution in 2-D gels was gradually improved with the increase of PVPP cleanup steps. By the protocol, about 760 protein spots of berry tissues were clearly resolved in 2-D gels with CBB staining. This protocol was also used to analyze β-1,3-glucanase (EC 3.2.1.39) in berry tissues. An anti-synthetic peptide antibody was prepared against 15 amino acid sequence residing on the surface of β-1,3-glucanase molecule. It detected two major spots in 2-D blots of berry extracts. The spots were identified by MALDI-TOF analysis as β-1,3-glucanase. The present study validates that β-1,3-glucanase is present in higher abundance in berry skins than in pulps, and in red berries than in white berries. Therefore, β-1,3-glucanase displays a tissue-specific expression. The preferential accumulation of β-1,3-glucanase in skins may be relevant to berry ripening.     

Identification of grapevine aquaporins and expression analysis in developing berries

Aquaporins are membrane water channels that play critical roles in controlling the water content of cells and tissues. In this work, nine full-length cDNAs encoding putative aquaporins were isolated from grape berry cDNA libraries. A phylogenetic analysis conducted with 28 aquaporin genes identified in the grapevine genome and previously characterized aquaporins from Arabidopsis indicates that three cDNAs encode putative tonoplast aquaporins (TIPs) whereas six cDNAs belong to the plasma membrane aquaporin subfamily (PIPs). Specific probes designed on the 3' untranslated regions of each cDNA were used for the preparation of cDNA macroarray filters and in situ hybridization experiments. Macroarray data indicate that expression levels of most TIP and PIP genes depend on grape berry developmental stages and point out to a global decrease of aquaporin gene expression during berry ripening. In young berries, high expression of aquaporin genes was preferentially observed in dividing and elongating cells and in cells involved in water and solutes transport. Taken together, the data provided in this paper indicate that aquaporins are implicated in various physiological aspects of grape berry development.